Systems, methods, and compositions for achieving closure of vascular puncture sites

ABSTRACT

Systems, methods, and compositions achieve rapid closure of vascular puncture sites. The systems and methods form a vascular closure composition by mixing together a first component, a second component, and a buffer material. The first component includes an electrophilic polymer material having a functionality of at least three. The second component includes a nucleophilic material that, when mixed with the first component within a reaction pH range of between 7 to 9, cross-links with the first component to form a non-liquid, three-dimensional barrier. The buffer material has a pH within the reaction pH range. The systems and methods apply the composition to seal a vascular puncture site.

RELATED APPLICATIONS

This application is a divisional of co-pending U.S. patent applicationSer. No. 09/780,014, filed Feb. 9, 2001, which is a continuation-in-partof U.S. patent application Ser. No. 09/283,535, filed Apr. 1, 1999, nowU.S. Pat. No. 6,458,147, which is itself a continuation-in-part of U.S.patent application Ser. No. 09/188,083, filed Nov. 6, 1998, now U.S.Pat. No. 6,371,975.

FIELD OF THE INVENTION

The invention generally relates to the composition of biocompatiblematerials and their application to body tissue to affect desiredtherapeutic results.

BACKGROUND OF THE INVENTION

There are many therapeutic indications today that pose problems in termsof technique, cost efficiency, or efficacy, or combinations thereof.

For example, following an interventional procedure, such as angioplastyor stent placement, a 5 Fr to 8 Fr arteriotomy remains. Typically, thebleeding from the arteriotomy is controlled through pressure applied byhand, by sandbag, or by C-clamp for at least 30 minutes. While pressurewill ultimately achieve hemostasis, the excessive use and cost of healthcare personnel is incongruent with managed care goals.

Various alternative methods for sealing a vascular puncture site havebeen tried. For example, collagen plugs have been used to occlude thepuncture orifice. The collagen plugs are intended to activate plateletsand accelerate the natural healing process. Holding the collagen sealsin place using an anchor located inside the artery has also been tried.Still, patient immobilization is required until clot formationstabilizes the site. Other problems, such as distal embolization of thecollagen, rebleeding, and the need for external pressure to achievehemostatis, also persist.

As another example, devices that surgically suture the puncture sitepercutaneously have also been used. The devices require the practice offine surgical skills to place needles at a precise distance from theedges of the puncture orifice and to form an array of suture knots,which are tightened and pushed from the skin surface to the artery wallwith a knot pusher, resulting in puncture edge apposition.

There remains a need for fast and straightforward mechanical andchemical systems and methods to close vascular puncture sites and toaccelerate the patient's return to ambulatory status without pain andprolonged immobilization.

There also remains a demand for biomaterials that improve the technique,cost efficiency, and efficacy of these and other therapeuticindications.

SUMMARY OF THE INVENTION

The invention provides systems, methods, and compositions for achievingclosure of vascular puncture sites. The systems and methods form avascular closure composition by mixing together a first component, asecond component, and a buffer material. The first component includeselectrophilic polymer material having a functionality of at least three.The second component includes a nucleophilic material that, when mixedwith the first component within a reaction pH range of between 7 to 9,cross-links with the first component to form a non-liquid,three-dimensional barrier. The buffer material has a pH within thereaction pH range. The systems and methods apply the composition to seala vascular puncture site.

In a preferred embodiment, the first component comprises poly(ethyleneglycol) having a molecular weight between 9,000 and 12,000. In a mostpreferred embodiment, the poly(ethylene glycol) has a molecular weightof 10,500±1500. In these preferred embodiments, the second componentcomprises human serum albumin at a concentration of about 25% or less,and the buffer material includes tris-hydroxymethylaminomethane. Thebuffer material can also include sodium carbonate anhydrous. In apreferred embodiment, the second component, when mixed with the buffermaterial, and prior to mixing with the first component, has a pH ofbetween 8.3 and 8.5.

Compositions that embody features of the invention are successful insealing femoral puncture sites in less than 40 seconds.

Features and advantages of the inventions are set forth in the followingDescription and Drawings, as well as in the appended claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a diagrammatic view of the basic formative components which,when mixed in a liquid state, cross-link to form a solid biocompatiblematerial composition that is well suited for closing a vascular puncturesite;

FIG. 2 is a schematic view of a system for delivering the basicformative components, while in a liquid state, to a vascular puncturesite;

FIG. 3 is a diagrammatic view of the resulting non-liquid,three-dimensional composition that forms after the formative components,shown in FIG. 1, cross-link to transform from a liquid state, to asemi-solid (gel) state, and then to a biocompatible solid state, whichcloses the vascular puncture site;

FIG. 4 is a perspective view of a catheter device that, when used in themanner shown in FIG. 2, delivers the basic formative components, whilein a liquid state, to a vascular puncture site;

FIG. 5 is a diagrammatic view showing operation of the catheter deviceshown in FIG. 4 to mix the basic formative components shown in FIG. 1,to deliver the components to a vascular puncture site, where theresulting cross-linking reaction affects closure to the vascularpuncture site; and

FIG. 6 is a diagrammatic view showing the temporary application oflocalized pressure to the skin surface as the formative componentscross-link to close the vascular puncture site.

The invention may be embodied in several forms without departing fromits spirit or essential characteristics. The scope of the invention isdefined in the appended claims, rather than in the specific descriptionpreceding them. All embodiments that fall within the meaning and rangeof equivalency of the claims are therefore intended to be embraced bythe claims.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

I. Overview FIG. 1 diagrammatically shows the basic formative components10, 12, and 14 of a solid biocompatible material composition 16, whichis shown in FIGS. 2 and 3 after the components 10, 12, and 14 have beenmixed. The composition 16 is well suited for closing a vascular puncturesite, e.g., following a vascular access procedure. The formativecomponents 10, 12, and 14 can be mixed in a liquid state while beingdelivered through a transcutaneous catheter 30 to the puncture site 32(see FIG. 2). Upon mixing, the formative components 10, 12, and 14 reactto transform in situ from the liquid state, to a semi-solid (gel) state,and then to the biocompatible solid state, in a process called“gelation.” In the solid state, the composition 16 takes the form of anon-liquid, three-dimensional network (as diagrammatically shown in FIG.3).

The solid material composition 16 exhibits desired mechanicalproperties. These properties include adhesive strength (adhering it toadjacent tissue), cohesive strength (forming a mechanical barrier thatis resistant to blood pressure and blood seepage), and elasticity(accommodating the normal stresses and strains of everyday activity).These properties, as well as the relative rapid rate of gelation thatcan be achieved, serve to provide a fast and effective closure to thevascular puncture site.

The solid material composition 16 is also capable of transforming overtime by physiological mechanisms from the solid state to a biocompatibleliquid state, which can be cleared by the body, in a process called“degradation.”

The time period that begins when the components 10, 12, and 14 have beenmixed and ends when the composition has reached the semi-solid (gel)state will be called the “gelation time.” When in this state, thecomposition possesses sufficient cohesive and adhesive strength toimpede blood flow, but still retains a self-sealing property, possessingthe capacity to close in upon and seal the tract left by the catheter inthe composition when the physician removes the catheter. For sealing avascular puncture site, the composition 16 preferably possesses agelation time that is in the range of fifteen to sixty seconds. Agelation time in the range of fifteen to thirty seconds is mostpreferred. This period allows the components 10, 12, and 14 forming thecomposition 16 to flow first in a liquid state, and then in thesemi-solid (gel) state, outward along the axis of the blood vessel. Theflow of components during gelation fills surface irregularities in thetissue region of the vascular puncture site 32, before solidificationoccurs. A gelation time period of between 10 and 40 seconds also fallswell within the time period a physician typically needs to manipulateand remove the catheter 30 after delivery of the components to thepuncture site 32. With an experienced physician, the cathetermanipulation and removal time period can be as quick as 10 to 40seconds, but it can extend, due to circumstances, upwards to 2 minutes.With a gelation time falling within the preferred range, the formationof the material composition 16 does not require a physician to “watchthe clock,” but rather attend only to the normal tasks of injecting thematerial and then manipulating and removing the catheter 30. With agelation time falling within the preferred range, it has been discoveredthat, if the catheter is removed in 15 seconds to 2 minutes followinginitial mixing, the composition 16 has reached a physical state capableof performing its intended function, while still accommodating a sealedwithdrawal of the catheter. Desirably, after removal of the catheter,the physician applies localized and temporary finger presssure to theskin surface above the composition for a period of about 5 minutes, toaid in the closure of the catheter tract in the composition, as thecomposition reaches its solid state.

The composition 16 preferably possesses sufficient adhesive strength toprevent dislodging from the arteriotomy, once formed. The composition 16also has sufficient cohesive strength to prevent rupture under arterialpressure, i.e., up to about 200 mm Hg. The composition 16 seals thearteriotomy for up to 15 days post-application before loss of mechanicalproperties through degradation, and degrades by 30 to 90 dayspost-application.

II. The Formative Components

The formative component 10 (see FIG. 1) comprises a solution 18containing an electrophilic (electrode withdrawing) material 20 having afunctionality of at least three. The formative component 12 comprises asolution 22 containing a nucleophilic (electron donator) material 24.When mixed under proper reaction conditions, the electrophilic material20 and nucleophilic material 24 react, by cross-linking with each other(as FIG. 5 diagrammatically shows). The cross-linking of the materials20 and 24 form the composition 16, which is diagrammatically shown inFIG. 3. The composition 16 physically forms a mechanical barrier, whichcan also be characterized as a hydrogel.

The formative component 14 comprises a solution 26 containing a buffermaterial 28. The type and concentration of the buffer material controlsthe pH of the components 10 and 12, when brought into contact formixing. The buffer material 14 desirably establishes an initial pH innumeric terms, as well regulates change of the pH over time (acharacteristic that will be called the “buffering capacity”).

The gelation time (which indicates the rate at which the cross-linkingreaction occurs) is controlled, inter alia, by the reaction pH, whichthe buffer component 14 establishes. The reaction pH controls thereactivity of nucleophilic groups in the second component 12, whichreact with the electrophilic groups in the first component 10. Generallyspeaking, the higher the reaction pH is, the larger is the fraction ofnucleophilic groups available for reaction with the electrophilicgroups, and vice versa.

To achieve a relatively rapid gelation time, a relatively high initialreaction pH (which, for the illustrated components, is above 8) isdesirable at the time initial mixing of the components 10, 12, and 14occurs. On the other hand, by the time the mixture is brought intocontact with body tissue at the vascular puncture site, it is desirablethat mixture possess a more physiologically tolerated pH level(approximately 7.4).

However, it has been discovered that, if the initial reaction pH is toohigh (which, for the illustrated components, is believed to be a pHapproaching about 9), the gelation time may be too rapid to consistentlyaccommodate the time period a physician typically requires to remove thecatheter, particularly if the time period approaches the two minutemark. In this instance, by the two minute mark, substantialsolidification of the composition can occur, and the composition canlack the cross-linking capacity to close in about the catheter tractleft in the composition upon removal of the catheter. Under thesecircumstances, blood leakage and hematoma formation can result afterremoval of the catheter.

Achieving and sustaining a reaction pH to meet a targeted gelation timeis therefor a critical criteria. It has been discovered that, bypurposeful selection of the components 10, 12, and 14, (i) an initiallyhigh reaction pH can be established that is conducive to rapid gelation,before contact with body tissue occurs, and (ii) the reaction pH can belowered as gelation progresses, as the mixture is delivered through thecatheter into contact with body tissue at the vascular puncture site. Atthe same time, by purposeful selection of the components 10, 12, and 14,the rate at which the pH is lowered during delivery can be mediated, sothat gelation is sustained at a rate that meets the gelation timerequirements to achieve the desired in situ formation of the composition16, one that also possesses sufficent cross-linking capacity to closeabout the catheter tract following removal of the catheter 30 after atime period a physician typically needs to perform this task.

In this context, the first component 10 desirably takes the form of asolution 18 of an electrophilic derivative of a hydrophilic polymer(which will also sometimes be referred generically as a “polymersolution.”). The second component 12 desirably takes the form of asolution of a material with nucleophilic groups (e.g., amines or thiols)(which will also sometimes be referred generically as a “proteinsolution.”).

The nucleophilic groups of the solution component 12 are also naturallypresent in the physiologic environment in which the composition 16forms. Thus, the same cross-linking reaction that occurs between theelectrophlic groups of the polymer solution and the nucleophilic groupsof the protein solution—building interior cohesive strength to thecomposition 16—will also occur between the electrophlic groups of thepolymer solution and the nucleophilic groups of body tissue—givingtissue adhesive strength to the composition 16 at the vessel puncturesite.

Furthermore, as the components 10 and 12 aggressively cross-link in afavorably high reaction pH environment, their cross-linking forms anon-toxic, acidic leaving group. Thus, as the gelation reaction proceedsat a high rate in a favorable high pH environment, the acidic leavinggroups that are formed will self-promote a decrease in the reaction pH,so that the pH of the mixture can be lowered to a more physiologicallytolerated pH level by the time it makes contact with body tissue. Oncecontact with body tissue occurs, the reaction pH will continue to bemediated by the surrounding pH (7.3 to 7.4) of the tissue region.

The rate of gelation will, of course, slow as the pH drops. To sustainan acceptable rate of gelation as the reaction environment becomes moreacidic, the type and concentration of the buffer material 28 areselected to provide a desired buffering capacity. The greater thebuffering capacity is, the greater is the ability of the bufferingmaterial 28 to absorb acidic groups as they form and mediate the rate atwhich the pH (and thus rate of gelation) drops, both before and afterentry into the body.

Further details of the most preferred, representative forms of thecomponents follow.

A. The Electrophilic Component

In its most preferred form, the electrophilic (electrode withdrawing)material 20 comprises a hydrophilic, biocompatible polymer that iselectrophilically derivatized with a functionality of at least three.Examples include poly(ethylene glycol), poly(ethylene oxide), poly(vinylalcohol), poly(vinylpyrrolidinone), poly(ethyloxazoline), andpoly(ethylene glycol)-co-poly(propylene glycol) block copolymers.

As used herein, a polymer meeting the above criteria is one that beginswith a multiple arm core (e.g., pentaerythritol) and not a bifunctionalstarting material, and which is synthesized to a desired molecularweight (by derivatizing the end groups), such that polymers withfunctional groups greater than or equal to three constitute (accordingto gel permeation chromatography-GPC) at least 50% or more of thepolymer blend.

The material 20 is not restricted to synthetic polymers, aspolysaccharides, carbohydrates, and proteins could be electrophilicallyderivatized with a functionality of at least three. In addition, hybridproteins with one or more substitutions, deletions, or additions in theprimary structure may be used as the material 20. In this arrangement,the protein's primary structure is not restricted to those found innature, as an amino acid sequence can be synthetically designed toachieve a particular structure and/or function and then incorporatedinto the material. The protein of the polymer material 20 can berecombinantly produced or collected from naturally occurring sources.

Preferably, the polymer material 20 is comprised of poly(ethyleneglycol) (PEG) with a molecular weight preferably between 9,000 and12,000, and most preferably 10,500±1500. PEG has been demonstrated to bebiocompatible and non-toxic in a variety of physiological applications.The preferred concentrations of the polymer are 5% to 35% w/w, morepreferably 5% to 20% w/w. The polymer can be dissolved in a variety ofsolutions, but sterile water is preferred.

The most preferred polymer material 20 can be generally expressed ascompounds of the formula:PEG−(DCR−CG)_(n)

-   -   Where;    -   DCR is a degradation control region.    -   CG is a cross-linking group.    -   n a 3.

The electrophilic CG is responsible for the cross-linking of thepreferred nucleophilic material 24, as well as binding the composition16 to the like material in the surrounding tissue, as will be describedlater. The CG can be selected to selectively react with thiols,selectively react with amines, or react with thiols and amines. CG'sthat are selective to thiols include vinyl sulfone, N-ethyl maleimide,iodoacetamide, and orthopyridyl disulfide. CG's that are selective toamines include aldehydes. Non-selective electrophilic groups includeactive esters, epoxides, oxycarbonylimidazole, nitrophenyl carbonates,tresylate, mesylate, tosylate, and isocyanate. The preferred CG's areactive esters, more preferred, an ester of N-hydroxysuccinimide. Theactive esters are preferred since they react rapidly with nucleophilicgroups and have a non-toxic leaving group, e.g., hydroxysuccinimide.

The concentration of the CG in the polymer material 20 can be used tocontrol the rate of gelation. However, changes in this concentrationtypically also result in changes in the desired mechanical properties ofthe hydrogel.

The rate of degradation is controlled by the degradation control region(DCR), the concentration of the CG's in the polymer solution, and theconcentration of the nucleophilic groups in the protein solution.Changes in these concentrations also typically result in changes in themechanical properties of the hydrogel, as well as the rate ofdegradation.

The rate of degradation (which desirably occurs in about 30 days) isbest controlled by the selection of the chemical moiety in thedegradation control region, DCR. If degradation is not desired, a DCRcan be selected to prevent biodegradation or the material can be createdwithout a DCR. However, if degradation is desired, a hydrolytically orenzymatically degradable DCR can be selected. Examples of hydrolyticallydegradable moieties include saturated di-acids, unsaturated di-acids,poly(glycolic acid), poly(DL-lactic acid), poly(L-lactic acid),poly(ξ-caprolactone), poly(δ-valerolactone), poly(γ-butyrolactone),poly(amino acids), poly(anhydrides), poly(orthoesters),poly(orthocarbonates), and poly(phosphoesters), and derivatives thereof.A preferred hydrolytically degradable DCR is gluturate. Examples ofenzymatically degradable DCR's include Leu-Gly-Pro-Ala (collagenasesensitive linkage) and Gly-Pro-Lys (plasmin sensitive linkage). Itshould also be appreciated that the DCR could contain combinations ofdegradable groups, e.g. poly(glycolic acid) and di-acid.

While the preferred polymer is a multi-armed structure, a linear polymerwith a functionality, or reactive groups per molecule, of at least threecan also be used. The utility of a given PEG polymer significantlyincreases when the functionality is increased to be greater than orequal to three. The observed incremental increase in functionalityoccurs when the functionality is increased from two to three, and againwhen the functionality is increased from three to four. Furtherincremental increases are minimal when the functionality exceeds aboutfour.

A preferred polymer may be purchased from Shearwater Polymers Inc.(Product Designation: PEG4SG, having a molecular weight range of between9000 and 12,000) (which will be called the “Shearwater PEG”). Gelpermeation chromatography of the Shearwater PEG reveals that (bymolecular weight) 59.2% of the Shearwater PEG comprises 4-Arm-PEGpolymer.

Alternatively, another preferred polymer may be purchased from SunBioCompany ((PEG-SG)4, having a molecular weight of 10,500±1500) (whichwill be called the “SunBio PEG”). Gel permeation chromatography of theSunBio PEG reveals that (by molecular weight) 3.1% of the SunBio PEGcomprises 3-Arm-PEG polymer and 90.7% of the SunBio PEG comprises4-Arm-PEG polymer. When compared to the Shearwater PEG, it can be seenthat the SunBio PEG contains a greater concentration of PEG polymerswith a functionality equal to or greater than 3.

B. The Nucleophilic Component

In a most preferred embodiment, the nucleophilic material 24 includesnon-immunogenic, hydrophilic proteins. Examples include serum, serumfractions, and solutions of albumin, gelatin, antibodies, fibrinogen,and serum proteins. In addition, water soluble derivatives ofhydrophobic proteins can be used. Examples include solutions ofcollagen, elastin, chitosan, and hyaluronic acid. In addition, hybridproteins with one or more substitutions, deletions, or additions in theprimary structure may be used.

Furthermore, the primary protein structure need not be restricted tothose found in nature. An amino acid sequence can be syntheticallydesigned to achieve a particular structure and/or function and thenincorporated into the nucleophilic material 24. The protein can berecombinantly produced or collected from naturally occurring sources.

The preferred protein solution is 25% human serum albumin, USP. Humanserum albumin is preferred due to its biocompatibility and its readyavailability.

The uses of PEG polymers with functionality of greater than threeprovides a surprising advantage when albumin is used as the nucleophilicmaterial 24. When cross-linked with higher functionality PEG polymers,the concentration of albumin can be reduced to 25% and below. Past usesof difunctional PEG polymers require concentrations of albumin wellabove 25%, e.g. 35% to 45%. Use of lower concentrations of albuminresult in superior tissue sealing properties with increased elasticity,a further desired result. Additionally, 25% human serum albumin, USP iscommercially available from several sources, however higherconcentrations of human serum albumin, USP are not commerciallyavailable. By using commercially available materials, the dialysis andultrafiltration of the albumin solution, as disclosed in the prior art,is eliminated, significantly reducing the cost and complexity of thepreparation of the albumin solution.

To minimize the liberation of heat during the cross-linking reaction,the concentration of the cross-linking groups of the fundamental polymercomponent is preferably kept less than 5% of the total mass of thereactive solution, and more preferably about 1% or less. The lowconcentration of the cross-linking group is also beneficial so that theamount of the leaving group is also minimized. In a typical clinicalapplication, about 50 mg of a non-toxic leaving group is produced duringthe cross-linking reaction, a further desired result. In a preferredembodiment, the CG comprising an N-hydroxysuccinimide ester hasdemonstrated ability to participate in the cross-linking reaction withalbumin without eliciting adverse immune responses in humans.

C. The Buffer Component

In the most preferred embodiment, a PEG reactive ester reacts with theamino groups of the albumin and other tissue proteins, with the releaseof N-hydroxysuccinimide and the formation of a link between the PEG andthe protein. When there are multiple reactive ester groups per PEGmolecule, and each protein has many reactive groups, a network of linksform, binding all the albumin molecules to each other and to adjacenttissue proteins.

This reaction with protein amino groups is not the only reaction thatthe PEG reactive ester can undergo. It can also react with water (i.e.,hydrolyze), thereby losing its ability to react with protein. For thisreason, the PEG reactive ester must be stored dry before use anddissolved under conditions where it does not hydrolyze rapidly. Thestorage container for the PEG material desirably is evacuated by use ofa vacuum, and the PEG material is stored therein under an inert gas,such as Argon or Nitrogen. Another method of packaging the PEG materialis to lyophilize the PEG material and store it under vacuum, or under aninert gas, such as Argon or Nitrogen. Lyophilization provides thebenefits of long term storage and product stability, as well as allowsrapid dissolution of the PEG material in water.

The conditions that speed up hydrolysis tend to parallel those thatspeed up the reaction with protein; namely, increased temperature;increased concentration; and increased pH (i.e., increased alkali). Inthe illustrated embodiment, temperature cannot be easily varied, sovarying the concentrations and the pH are the primary methods ofcontrol.

It is the purpose of the buffer material 28 to establish an initial pHto achieve a desired gelation time, and to sustain the pH as added acidis produced by the release of N-hydroxysuccinimide during cross linkingand hydrolysis.

pH is the special scale of measurement established to define theconcentration in water of acid (H+) or alkali (OH—) (which, strictlyspeaking, indicates hydrogen ion activity). In the pH scale, solutionsof acid (H+) in water have a low pH, neutrality is around pH 7, andsolutions of base (OH—) in water have a high pH. The pH scale islogarithmic. A change of one pH unit (e.g., from pH 9 to pH 10)corresponds to a ten-fold change in concentration (i.e., hydrogen ionactivity). Thus, reactions which are increased by alkali, such ashydrolysis of PEG reactive ester, are expected to increase in rate by afactor of ten for each unit increase in pH.

The buffer material 28 is a mixture of molecules, added to the albumin,that can moderate pH changes by reacting reversibly with added acid (H+)or base (OH—). The pH moderating effect can be measured by titration,i.e., by adding increasing amounts of H+ or OH— to the buffer material,measuring the pH at each step, and comparing the pH changes to that of asimilar solution without the buffer.

Different buffers exert a maximum pH moderating effect (i.e., the leastchange in pH with added H+ or OH—) at different pH's. The pH at which agiven buffer exerts its maximum pH moderating effect is called its pK.

Even when the pH matches the pK for a given buffer, added acid or basewill produce some change in pH. As the pH changes from the pK value, themoderating effect of the buffer decreases progressively (e.g., 67% lessat +/−1 pH unit from pK, and 90% less at +/−1.6 pH unit from pK). Themoderating effect is also proportional to the buffer concentration.Thus, increasing the buffer concentration increases the ability tomoderate pH changes.

The overall buffering effect at any pH is the sum of all bufferingspecies present, and has also been earlier called the bufferingcapacity. The higher the buffering capacity, the more acid or base mustbe added to produce a given pH change. Stated differently, the higherthe buffering capacity, the longer a given buffer is able to sustain atargeted pH range as acid or base is being added to change the pH.

Albumin itself contains amino, carboxyl, and other groups, which canreversibly react with acid and base. That is, albumin itself is abuffer. Also, due to the many different buffering groups that albuminpossesses, albumin (e.g., Plasbumin) can buffer over a relatively broadpH range, from below pH 6 to over pH 10. However, it has been discoveredthat albumin lacks the buffering capacity to, by itself, counterbalancethe additional acid (H+) that is produced as a result of hydrolysis andthe PEG-albumin cross-linking, given the PEG concentrations required tomeet the therapeutic objectives for the composition. Thus, in thepreferred embodiment, a buffer material 28 must be added to the albuminto provide the required buffering capacity.

The buffer material 28 must meet several criteria. The buffer materialmust be (1) non-toxic, (2) biocompatible, (3) possess a pK capable ofbuffering in the pH range where the desirable gelation time exists, and(4) must not interfere with the reaction of protein with the selectedPEG reactive ester. Amine-containing buffers do not meet criteria (4).

To meet criteria (3), the buffer material 28 should have a bufferingcapacity at the desired cross-linking pH (i.e., as indicated by its pK)that is high enough to counterbalance the additional acid (H+) producedas a result of the cross-linking reaction and hydrolysis, i.e., to keepthe pH high enough to achieve the desired gelation time.

It has been discovered, through bench testing, that when cross-linkingthe Shearwater PEG and SunBio PEG with albumin (Plasbumin), a range ofgelation times between an acceptable moderate time (about 30 seconds) toa rapid time (about 2 seconds) can be achieved by establishing a pHrange from about 8 (the moderate times) to about 10 (the rapid times).Ascertaining the cross-linking pH range aids in the selection of buffermaterials having pK's that can provide the requisite buffering capacitywithin the pH range.

Phosphate, tris-hydroxymethylaminomethane (Tris), and carbonate are allnon-toxic, biocompatible buffers, thereby meeting criteria (1) and (2).Phosphate has a pK of about 7, which provides increased bufferingcapacity to albumin at pH's up to about 8.5. Tris has a pK of about 8,which provides increased buffering capacity to albumin at pH's up toabout 9.5. Addition of Tris to albumin (Plasbumin) at a concentration of60 mM approximately doubles the buffering capacity of the albumin at apH near 9. Carbonate has a pK of about 10, and provides increasedbuffering capacity to albumin in the higher pH ranges. Depending uponthe gellation time that is targeted, formulations of Tris, carbonate,and albumin can be used for the buffer material 24.

EXAMPLE 1 Sodium Carbonate/Sodium Phosphate Buffer Formulations

Albumin (Human 25%, Plasbumin®-25 manufactured by Bayer Corporation) wasbuffered using differing amounts of Sodium Carbonate Monohydrate(Na₂CO₃H₂O) (FW 124.00) (“Carbonate Buffer”) and Sodium PhosphateMonobasic Monohydrate (NaH₂PO₄H₂O) (FW 137.99) (“Phosphate Buffer”). Thebuffered albumin formulations (2 cc) were mixed with 2 cc of theShearwater PEG (0.45 g of PEG suspended in 2.2 cc of water), to provide17% w/w PEG solids. The components were mixed by using the catheterdevice as generally shown in FIG. 2 (and as will be described in greaterdetail later). Upon mixing, the components were deposited in a testdish. The pH of the buffered albumin formulation (albumin plus buffermaterial) and the gelation time (as described above) and were recorded.TABLE 1 Albumin Carbonate Phosphate Device Gelling (Human 25%) BufferBuffer (Outside Time (ml) (grams) (grams) pH Diameter) (Seconds) 500.375 0.275 8.0   8 Fr 70 50 0.375 0.275 8.0 5.5 Fr 70 50 0.60 0.275 9.0  8 Fr 30 50 0.95 0.275 9.5   8 Fr 15

Table 1 shows that increasing the amount of Carbonate Buffer (pK 10)increases the pH and likewise reduces the gelation time. However,gelation times falling within the desired range required higher, lessdesirable pH's.

EXAMPLE 2 Carbonate Buffer/Tris Buffer Formulations

Albumin (Human 25%, Plasbumin®-25 manufactured by Bayer Corporation) wasbuffered using Sodium Carbonate Anhydrous (Na₂CO₃) (FW 106.0)(“Carbonate Buffer”) mixed with Tris-hydroxymethylaminomethane(C₄H₁₁NO₃) (FW 121.1) (“Tris Buffer”). The buffered albumin formulations(2 cc) were mixed with 2 cc of the Shearwater PEG (0.45 g of PEGsuspended in 2.2 cc of water), to provide 17% w/w PEG solids, in thesame manner as Example 1. The pH of the buffered albumin formulation(albumin plus buffer material) and the gelation time (as describedabove) and were recorded.

Table 2 summarizes the results. TABLE 2 Albumin Carbonate Tris DeviceGelling (Human 25%) Buffer Buffer (Outside Time (ml) (grams) (grams) pHDiameter) (Seconds) 20 0.137 0.145 9.0 8 Fr 15-16 20 0.137 0.145 9.0 8Fr 15-16

Table 2 shows faster gelation times, compared to the gelation times inTable 1, despite the presence of less Carbonate Buffer (pK 10). The morerapid gelling times are due to the increased buffering capacity that theTris Buffer (pK 8) provides in the pH range 7 to 9. In comparison, theCarbonate Buffer has little buffer capacity in the pH 7 to 9 range,being more effective at higher pH's. The higher pH levels (above aboutpH 9) are not desirable, as the composition is not quickly neutralizedby blood contact to terminate gelation (at about pH 7.4), should leakageof the composition into a blood path occur. Better buffering capacitythat the Tris Buffer provides in the pH 7 to 9 range keeps the pH fromdropping as quickly, which, in turns, consistently leads to a gelationtime falling within the desired range and at lower pH levels. The lowerpH levels (below about pH 9) are desirable because the composition ismore quickly neutralized by blood contact (at about pH 7.4), so that thepotential for gellation within the blood path is minimized.

EXAMPLE 3 Carbonate Buffer/Tris Buffer Formulations

Albumin (Human 25%, Plasbumin®-25 manufactured by Bayer Corporation) wasbuffered using Sodium Carbonate Anhydrous (Na₂CO₃) (FW 106.0)(“Carbonate Buffer”) mixed with Tris-hydroxymethylaminomethane(C₄H₁₁NO₃) (FW 121.1) (“Tris Buffer”). The buffered albumin formulations(2 cc) were mixed with 2 cc of the SunBio PEG (0.45 g of PEG suspendedin 2.2 cc of water), to provide 17% w/w PEG solids. The components weremixed in the manner described in Example 1. The pH of the bufferedalbumin formulation (albumin plus buffer material) and the gelation time(as described above) and were recorded.

Table 3 summarizes the results. TABLE 3 Albumin Carbonate Tris DeviceGelling (Human 25%) Buffer Buffer (Outside Time (ml) (grams) (grams) pHDiameter) (Seconds) 20 0 0.217 8.3 7 Fr 11 20 0 0.290 8.5 7 Fr  7-8 200.075 0.145 8.7 7 Fr  5-6 20 0.138 0.145 9.0 7 Fr  2-3

Table 3 shows even faster gelation times, compared to the gelation timesin Tables 1 and 2, at lower pH's. This is believed due to the largerconcentration of multiple functionality PEG in the SunBio PEG, as wellas the enhanced buffering capacity that the Tris Buffer (pK 8) providesin the lower pH range (7 to 9). It is also believed that the gelationtime will also vary, given the same composition, according to the sizeand configuration of the delivery device. The addition of CarbonateBuffer (in the pH 8.7 and pH 9 compositions) leads to a further decreasein gelation time, at an increased pH.

Tests of pH 8.3 and pH 8.5 compositions in Table 3 have demonstratedthat both composition are successful in sealing femoral puncture sitesin sheep in 25 to 40 seconds. The tests also show that eithercomposition possesses sufficient cross-linking capacity to close aboutthe catheter tract following removal of the catheter upwards to twominutes after delivery of the material. Both compositions therebyreadily accommodate variations in procedure time.

Tests of pH 8.7 composition in Table 3 have also demonstrated that thecomposition is successful in sealing femoral puncture sites in sheep in25 to 40 seconds. The tests also show that, due to the more rapidgelation time, the composition does not possesses sufficientcross-linking capacity to consistently close about the catheter tractfollowing removal of the catheter two minutes after delivery of thematerial. In this respect, the pH 8.7 composition, despite its fastergelation time, is not as accommodating to changes in procedure time asthe pH 8.3 and pH 8.5 compositions, described above. For these reasons,the most preferred range for vessel puncture sealing is between pH 8.3and pH 8.5.

II. Delivering the Formative Components to Create the Composition toClose Vascular Puncture Sites

Generally speaking, there are four stages in creating the composition 16to close a given vascular puncture site. These stages are: (1) theintroduction stage; (2) the localized compression stage; (3) thehemostasis stage; and (4) the degradation stage.

The phase of the composition 16 differs in each stage, as differentphysical and physiological events unfold. These different compositionphases are, respectively: (1) the liquid phase; (2) the semi-solid (gel)phase; (3) the solid phase; and (4) the re-absorption phase.

A. The Introduction Stage (The Composition Liquid Phase)

In the first stage, the physician introduces the catheter 30 through atissue track 34 to the site of the vascular puncture 32.

Typically, the catheter 30 is introduced along a guide wire 36 partiallyinto the blood vessel (see FIG. 2). The guide wire 36 will have beenpreviously introduced subcutaneously, through a wall of the bloodvessel, to guide passage of a desired therapeutic or diagnosticinstrument into the blood vessel, e.g., to perform coronary angioplasty.After performing the intended procedure, the therapeutic or diagnosticinstrument is withdrawn, leaving the guide wire 36. The catheter 30 isintroduced along the guide wire 36 to the puncture site 32. For reasonsthat will be explained in greater detail later, the diameter of thecatheter 30 is preferably sized to seal, but not enlarge, the tissuetrack 34.

In the illustrated embodiment (see FIGS. 2 and 4), the distal end of thecatheter 30 carries a balloon 38. When the balloon 38 is expanded withinthe blood vessel (as FIG. 2 shows), back pressure on the catheter 30serves to locate (by tactile pressure) the distal nozzles 40 outside thepuncture site 32 (see FIG. 4). The composition 16 is introduced throughthe nozzles 40.

A suitable material introducer/mixing device 44 is coupled to the distalend of the catheter 30. The device 44 can take various forms. In theillustrated embodiment (see FIG. 4), the device 44 includes a syringesupport 46, which receives, e.g., in a snap-fit, two dispensing syringes60 and 62. The pistons of the syringes 60 and 62 can be joined by asyringe clip 48. The clip 48 mechanically links the syringe pistonstogether for common advancement inside their respective syringe barrels.

The device 44 also includes a joiner 50 that includes two interiorchannels 52 and 54 (see FIG. 5). One channel 52 communicates with thesyringe 60, and the other channel 54 communicates with the syringe 62.The channels merge at a Y-junction 56 into a single outlet port.

The joiner 50 maintains the two fluids dispensed by the syringes 60 and62 separately until they leave the joiner 50. The outlet portcommunicates with an interior lumen in the catheter 30, which extends tothe nozzles 40.

The syringe 60 contains the electrophilic component 10 suspended insolution in sterile water, e.g., 17% w/w 4-arm poly(ethylene glycol)tetra-succinimidyl glutarate, MW 10,500±1500 in water for injection(most preferably, the SunBio PEG, as decribed in Example 3).

The syringe 62 carries the nucleophilic component 12 suspended insolution, along with the buffer component 14, e.g., 25% w/w human serumalbumin, USP supplemented with the Tris Buffer as shown in Table 3. ThepH 8.3 and pH 8.5 compositions in Table 3 are most preferred.

Operation of the device 44 expresses the components 10, 12, and 14,while in liquid form, from the syringes 60 and 62. As FIG. 5 shows, theliquid components 10, 12, and 14 begin to mix at the junction 56. Here,the initially high reaction pH is established, to begin thecross-linking reaction at a desired high rate. Mixing and attendantgelation continue, as the components 10, 12, and 14 proceed in responseto pressure exerted by the device 44 down the catheter lumen toward thenozzles 40.

Concurrently, the formation of the acidic leaving groups serves toreduce the reaction pH. By the time the components 10, 12, and 14, whichare still undergoing gelation, exit the nozzles 40, the reaction pH isapproaching a more physiologically tolerated level. Once in contact withbody tissue, the prevailing physiologic pH levels further mediate thereaction pH.

The gelating components 10, 12, and 14 flow out the nozzles 40 and intothe surrounding tissue mass (as FIG. 5 shows). The catheter 30, which issized to seal the tissue track 34, blocks substantial flow in a path upthe tissue track 34. Thus, the gelating components are directed in aflow radially away from the axis of the catheter 30 and along the axisof the vessel 42, as FIG. 5 shows.

The size of the catheter is selected according to the outside diameterof the introducer sheath used during the preceding therapeutic ordiagnostic procedure, during which the arteriotomy was made. Forexample, a 6 Fr introducer sheath typically has an outside diameter of 7Fr, so a 7 Fr diameter catheter is selected to seal the arteriotomy uponremoval of the introducer sheath. The composition 16 is delivered in aliquid state adjacent to the arteriotomy, while the catheter 30 preventsthe liquid from filling the tissue track. This feature ensures that thematerial composition 16 remains at the arteriotomy for maximum efficacy.The incoming flow, directed in this manner, creates a tissue space aboutthe puncture site along the axis of the vessel. The gelating componentsfill this space.

In the gelation process, the electrophilic component 10 and thenucleophilic component 12 cross-link, and the developing composition 16gains cohesive strength to close the puncture site. The electrophiliccomponent 10 also begins to cross-link with nucleophilic groups on thesurrounding tissue mass. Adhesive strength forms, which begins to adherethe developing composition 16 to the surrounding tissue mass.

During the gelation period, before internal cohesive and tissue adhesivestrengths fully develop, a portion of the gelating components 10, 12, 14can enter the blood vessel 42 through the puncture site 32. Uponentering the blood stream, the gelating components will immediatelyexperience physical dilution. The dilution expands the distance betweenthe electrophilic component 10 and the nucleophilic component 12, makingcross-linking difficult. In addition, the diluted components nowexperience an environment having a pH (7.3 to 7.4) lower than the aneffective reactive pH for cross-linking (which is above 8). As a result,incidence of cross-linking within the blood vessel, to form the hydrogelcomposition 16, is only a fraction of what it is outside the vessel,where gelation continues.

Furthermore, the diluted electrophilic component 10 will absorbnucleophilic proteins present in the blood. This reaction furtherreduces the reactivity of the electrophilic component 10. In blood, thediluted electrophilic component 10 is transformed into a biocompatible,non-reactive entity, which can be readily cleared by the kidneys andexcreted. The diluted nucleophilic component 12 is a naturally occurringprotein that is handled in normal ways by the body.

The Introduction Stage (The Composition Liquid Phase) preferably lastabout 5 to 30 seconds from the time the physician begins to mix thecomponents 10, 12, and 14.

B. The Localized Compression Stage (The Semi-Solid Composition Phase)

The second stage begins after the physician has delivered the entireprescribed volume of components 10, 12, and 14 to the tissue mass of thevessel puncture site and allowed the cross-linking of the components toprogress to the point where a semi-solid gel occupies the formed tissuespace.

At this point, the physician collapses the balloon 38 and withdraws thecatheter 30 and guide wire 36 from the tissue track 34. As FIG. 6 shows,the physician now simultaneously applies localized and temporarycompression to the skin surface surrounding the tissue track 34.

The application of localized pressure serves two purposes. It is not toprevent blood flow through the tissue track 34, as cross-linking of thecomponents has already proceeded to create a semi-solid gel havingsufficient cohesive and adhesive strength to impede blood flow from thepuncture site 32. Rather, the localized pressure serves to compress thetissue mass about the semi-solid gel mass. This compression brings thesemi-solid gel mass into intimate contact with surrounding tissue mass,while the final stages of cross-linking and gelation take place.

Under localized compression pressure, the electrophilic component 10 andthe nucleophilic component 12 are placed in close proximity, to completetheir cross-linking and to fully develop cohesive strength. Any remnantcatheter track existing through the gel mass will also be closed in thisprocess.

Under localized compression pressure, surface contact betweenelectrophilic component 10 and tissue is also increased, to promote thecross-linking reaction with nucleophilic groups in the surroundingtissue mass. Adhesive strength between the gel mass and tissue isthereby allowed to fully develop, to firmly adhere the gel mass to thesurrounding tissue as the solid composition 16 forms in situ.

During this stage, blood will also contact the vessel-side, exposedportion of the gel mass, which now covers the tissue puncture site 32.The electrophilic component 10 will absorb nucleophilic proteins presentin the blood, forming a biocompatible surface on the inside of thevessel.

The Localized Compression Stage (The Composition Semi-Solid (Gel) Phase)preferably last about 10 to 40 seconds from the time the physicianwithdraws the catheter 30.

C. The Hemostasis Stage (The Composition Solid Stage)

At the end of the Localized Compression Stage (see FIG. 6), thecomposition 16 has formed. Hemostasis has been achieved. The individualis free to ambulate and perform normal day-to-day functions.

The mechanical properties of the composition 16 are such to form amechanical barrier. The composition 16 is well tolerated by the body,without invoking a severe foreign body response.

The mechanical properties of the hydrogel are controlled, in part, bythe number of crosslinks in the hydrogel network as well as the distancebetween crosslinks. Both the number of crosslinks and the distancebetween crosslinks are dependent on the functionality, concentration,and molecular weight of the polymer and the protein.

Functionality, or the number of reactive groups per molecule, affectsthe mechanical properties of the resulting hydrogel by influencing boththe number of and distance between crosslinks. As discussed previously,the utility of a given polymer significantly increases when thefunctionality is increased to be greater than or equal to three. Theobserved incremental increase in functionality occurs when thefunctionality is increased from two to three, and again when thefunctionality is increased from three to four. By increasing thefunctionality of the polymer or protein at a constant concentration, theconcentration of crosslinking groups available for reaction areincreased and more crosslinks are formed. However, increased mechanicalproperties cannot be controlled with functionality alone. Ultimately,the steric hindrances of the protein or polymer to which the reactivegroups are attached predominate and further changes in the mechanicalproperties of the hydrogel are not observed. The effect of functionalityis saturated when the functionality reaches about four.

The concentration of the protein and polymer also affect the mechanicalproperties of the resulting hydrogel by influencing both the number ofand distance between crosslinks. Increasing the protein and polymerconcentration increases the number of available crosslinking groups,thereby increasing the strength of the hydrogel. However, decreases inthe elasticity of the hydrogel are observed as the concentration of theprotein and polymer is increased. The effects on the mechanicalproperties by concentration are limited by the solubility of the proteinand polymer.

The polymer and protein molecular weight affects the mechanicalproperties of the resulting hydrogel by influencing both the number ofand distance between crosslinks. Increasing the molecular weight of theprotein and polymer decreases the number of available crosslinkinggroups, thereby decreasing the strength of the hydrogel. However,increases in the elasticity of the hydrogel are observed with increasingmolecular weight of the protein and polymer. Low molecular weightproteins and polymers result in hydrogels that are strong, but brittle.Higher molecular weight proteins and polymers result in weaker, but moreelastic gels. The effects on the mechanical properties by molecularweight are limited by the solubility of the protein and polymer.However, consideration to the ability of the body to eliminate thepolymer should be made, as large molecular weight polymers are difficultto clear.

D. The Degradation Stage (The Composition Re-Absorption Stage)

Over a controlled period, the material composition 16 is degraded byphysiological mechanisms. Histological studies have shown a foreign bodyresponse consistent with a biodegradable material, such as VICRYL™sutures. As the material is degraded, the tissue returns to a quiescentstate. The molecules of the degraded genus hydrogel composition arecleared from the bloodstream by the kidneys and eliminated from the bodyin the urine. In a preferred embodiment of the invention, the materialloses its physical strength during the first fifteen days, and totallyresorbs in about four weeks.

The features of the invention are set forth in the following claims.

1. A method for sealing a vascular puncture site comprising the steps ofmixing a first component, a second component, and a buffer material, thefirst component including an electrophilic polymer material comprisingpoly(ethylene glycol) having a molecular weight between 9000 and 12,000and having a functionality of at least three, the second componentincluding a nucleophilic material comprising a protein at aconcentration of about 25% or less that, when mixed with the firstcomponent within a reaction pH range of between 7 to 9, cross-links withthe first component to form a non-liquid, three-dimensional barrier, andthe buffer material including tris-hydroxymethylaminomethane having a pHwithin the reaction pH range, and applying the mixture to seal avascular puncture site.
 2. A method according to claim 1 wherein thepoly(ethylene glycol) has a molecular weight of 10,500±1500.
 3. A methodaccording to claim 1 wherein the buffer material includes sodiumcarbonate anhydrous.
 4. A method according to claim 3 wherein the secondcomponent, when mixed with the buffer material, has a pH of between 8.3and 8.5 prior to mixing with the first component.